ABSTRACT ?? T cells mediate adaptive immune recognition essential for self vs. non-self discrimination via clonally distributed T cell receptors (TCRs) generated in the thymus through ? and ? gene rearrangements. Sculpting of the T cell repertoire through both positive and negative selection occurs at the CD4+CD8+ double positive (DP) stage of thymic development where ?? TCRs first appear. Prior to the DP stage, individual ? chains associate with the invariant pre-T? (pT?), forming preTCRs on the surface of early thymocytes. pT? consists of a C?-like Ig domain lacking a V domain. Signaling through preTCR was considered a ligand-independent, autonomous process. Our recent data show otherwise. Using NMR spectroscopy, interactions between two distinct preTCR ? chains and peptide/MHC (pMHC) ligands are observed involving canonical V? CDR loops and a hydrophobic patch accessible on the molecular surface of the unpaired preTCR V? domain. These observations have been recapitulated and extended using biomembrane force probe (BFP) and optical tweezers (OT) analyses of preTCRs on intact early thymocytes. Mutation of preTCR V? CDR and patch residues impacts early thymocyte proliferation and developmental progression to the DP stage in both fetal thymic organ culture (FTOC) and OP9- DL4 stromal cell-dependent differentiation systems. PreTCR-pMHC interaction triggers thymocyte calcium flux, revealing ligand-dependent signaling. Here we pursue three aims to define preTCR mechanobiology. In Aim 1, we will use high throughput next generation sequencing (NGS) of thymocyte subpopulations to determine ? repertoire changes as a consequence of preTCR-ligand interaction in vitro using single chain pMHC expressing stroma and thymocyte progenitors as well as by performing in vivo analysis in B6 MHC sufficient and deficient mice. We shall identify ? chains selected for loss or survival during thymic development and perform RNAseq on B6 Rag2-/- thymocytes transduced with each type, determining the relationship of preTCR ligation, biomechanics and transcriptome at population and single cell level. In Aim 2, structural features and mechanobiology of the preTCRs will be interrogated with single molecule (SM) OT experiments to assess the impact of diverse preTCRs on structural transitions, bond lifetimes, hopping between compact and extended states and their relationship to thymic development. In addition, structural studies by NMR in collaboration with Project 3, Core B, Core C and X-ray crystallography shall define individual molecular population states of ligated and unligated preTCR conformers. In Aim 3, we will determine if ??TCRs, distinct from ??TCRs and preTCRs in lacking the elongated C? chain FG loop element, manifest bond prolongation under force. Both human and mouse ??TCRs with defined CD1c or CD1d ligand specificities as well as G8 ?? that interacts with T22 will be compared using SM, single molecule single cell (SMSC) and single cell (SC) OT analyses with chimeric versions in which V?V? module is intact but C?C? replaces the C?C? constant module. B6 Rag2-/- thymocyte transduced wild type ?? versus chimeric TCRs will be assessed for progression in the presence or absence of ligands.